Use of siRNA in the preparation of TILRR expression inhibitors

Abstract

The application relates to application of siRNA in preparation of TILRR expression inhibitors, a target sequence interfered by the siRNA includes a nucleic acid sequence shown in SEQ ID NO. 1 and / or SEQ ID NO. 2, and a nucleic acid sequence of the siRNA includes a sequence shown in SEQ ID NO. 3 and / or SEQ ID NO. 4. The application takes TILRR as a treatment target, reduces TILRR expression by using siRNA, reduces the response of cells to inflammatory stimulation, does not completely block the inflammatory signal pathway, and reduces side effects caused by treatment by blocking an innate immune receptor in different inflammation-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of siRNA in preparation of TILRR expression inhibitor. BACKGROUND

[0002] The TIR (Toll-like receptor and Interleukin-1 receptor) family contains two groups of transmembrane proteins that share functional and structural properties, including the Interleukin-1 receptor (IL-1R) subfamily and the Toll-like receptor (TLR) subfamily. The hallmark of the TIR family is the cytoplasmic TIR domain that is essential for signal transduction, which can serve as a scaffold for a series of protein interactions, activating the signaling pathways such as NF-κB by a signaling module consisting of MyD88, Interleukin-1 receptor-associated kinase (IRAK) family members and Tollip, leading to inflammatory responses. TLR4 is the receptor for major lipopolysaccharide (LPS) and some DAMP molecules, which induces the release of pro-inflammatory factors such as TNF-α, IL-6 and IL-1β by interacting with LPS / DAMP molecules, and IL-1R1 is the receptor for IL-1β. The activation of TLR4 and IL-1R1 receptors induces the release of pro-inflammatory factors such as TNF-α, IL-6 and IL-1β, which play a core role in inflammatory responses. The signaling mechanisms of TLR4 and IL-1R1 are important targets for studying inflammatory diseases, and they play a key role in infection, autoimmune diseases and chronic inflammation.

[0003] However, direct targeting of inflammatory factors or receptors has certain side effects. For example, IL-1R1, as the receptor for inflammatory factor IL-1β, has a "double-edged sword" effect in injury repair. Knocking out IL-1R1 can reduce the pathological process of atherosclerotic plaques, but also enhances the instability of atherosclerotic plaques. In kidney injury, knocking out IL-1R1 in proximal tubular cells can shorten the disease process of aristolochic acid (AA)-induced kidney injury, while knocking out IL-1R1 specifically in endothelial cells shows an exacerbation of the disease process of AA-induced kidney injury. Further analysis of the mechanism shows that specific knockout of IL-1R1 in proximal tubular and endothelial cells does not affect the inflammatory response after AA-induced kidney injury, and it is found in proximal tubular cells that cell damage and death caused by activation of IL-1R1 are related to lipid metabolism. Therefore, directly targeting innate immune receptors as targets for treating inflammation-related diseases has certain effects, but there is a risk of side effects.

[0004] Toll-like receptor / IL-1 receptor regulator (TILRR, Frem1 isoform 2) interacts with IL-1R1, increases the recruitment of MYD88 to the intracellular TIR domain of IL-1R1, and thus increases the downstream NF-κB signal flow and promotes the expression of related pro-inflammatory factors. Knocking out TILRR or blocking the interaction between TILRR and IL-1R1 not only reduces the pathological process of atherosclerotic plaques, but also increases the stability of the plaques. Further studies have found that overexpression of TILRR can increase the response of cells to LPS stimulation. Therefore, TILRR is used as a therapeutic target to reduce inflammation and reduce the risk of side effects caused by blocking innate immune receptors.

[0005] siRNA (small interfering RNA) is a double-stranded RNA molecule composed of 20-25 nucleotides, which mainly plays a role in the RNA interference (RNAi) pathway, and inhibits or closes the expression of specific genes by specifically degrading homologous mRNA. siRNA is incorporated into the RNA-induced silencing complex (RISC). In RISC, the siRNA double strand is opened, the reverse strand is degraded, and the guide strand remains in RISC. The guide strand complementary to the target mRNA guides the RISC complex to the mRNA, and the nuclease (such as Argonaute protein) in the RISC complex degrades the target mRNA, preventing it from being translated into protein, thereby achieving gene silencing. Due to its high specificity and effectiveness, siRNA technology has very important application value in gene function research, disease treatment and new drug development. Targeting the mRNA of a specific gene through siRNA can be used to treat various diseases including tumors, infectious diseases, genetic diseases, etc., and therefore has been widely concerned in the field of life sciences and is considered as a highly potential direction in the field of future new drug development.

[0006] In summary, how to reduce the response of cells to inflammatory stimuli without completely blocking the inflammatory signaling pathway is one of the problems to be solved in the art. SUMMARY

[0007] To solve the above technical problems, the present application provides the use of siRNA in the preparation of a TILRR expression inhibitor, which uses siRNA to achieve TILRR gene silencing, thereby reducing the response of cells to inflammatory stimuli.

[0008] To achieve this purpose, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides the use of siRNA in the preparation of a TILRR expression inhibitor, wherein the target sequence interfered by the siRNA comprises the nucleic acid sequence shown in SEQ ID NO. 1 and / or SEQ ID NO. 2.

[0010] SEQ ID NO. 1: GAAGUGGUCUCAUAUUGAAUG.

[0011] SEQ ID NO. 2: CCAGGAAUGUCAACUAAGAUG.

[0012] Currently, the therapeutic effect of diseases mediated by innate immune inflammatory signals such as interleukin 1 and lipopolysaccharide is mainly achieved by targeting inflammatory factors / innate immune receptors, but this treatment strategy can completely block the innate immune related signal pathway. These treatment methods have certain effects, but often have side effects. The present application reduces the expression of TILRR by siRNA, reduces the response of cells to inflammatory stimulation, and does not completely block the inflammatory signal pathway, which can avoid the side effects of completely blocking the innate immune related signal pathway. The TILRR RNA target sequence provided by the present application has an appropriate GC content, and the siRNA targeting the target sequence has high efficiency in reducing the expression of TILRR gene.

[0013] Preferably, the nucleic acid sequence of the siRNA comprises the sequence shown in SEQ ID NO. 3 and / or SEQ ID NO. 4.

[0014] SEQ ID NO. 3: CAUUCAAUAUGAGACCACUUCTT.

[0015] SEQ ID NO. 4: CAUCUUAGUUGACAUUCCUGGTT.

[0016] Preferably, the target sequence interfered by the siRNA is the nucleic acid sequence shown in SEQ ID NO. 1, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO. 3.

[0017] Preferably, the target sequence interfered by the siRNA is the nucleic acid sequence shown in SEQ ID NO. 2, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO. 4.

[0018] In a second aspect, the present application provides a use of a TILRR expression inhibitor in the preparation of a preparation for inhibiting the expression of pro-inflammatory factors.

[0019] Preferably, the TILRR expression inhibitor comprises any one or a combination of at least two of a nucleic acid molecule, a nucleic acid construct, a lentivirus, an antibody, or a small molecule compound.

[0020] Preferably, the nucleic acid molecule comprises any one or a combination of at least two of double-stranded RNA, siRNA, or shRNA.

[0021] Preferably, the nucleic acid sequence of the siRNA comprises the sequence shown in SEQ ID NO. 3 and / or SEQ ID NO. 4.

[0022] Preferably, the source of the pro-inflammatory factor comprises stimulation of cells or organs using exogenous or endogenous inflammatory factors.

[0023] Preferably, the inflammatory factor comprises IL-1β and / or lipopolysaccharide.

[0024] Preferably, the pro-inflammatory factor comprises any one or a combination of at least two of CXCL8, TNF or CCL2.

[0025] The present application is designed to use specific TILRR expression inhibitors to effectively inhibit the expression of pro-inflammatory factors, and the TILRR expression inhibitors can be used as drugs for inhibiting the expression of pro-inflammatory factors, and can also be used for basic research or construction of experimental models (cells / animals / organoids / microphysiological models) for non-disease treatment purposes, etc.

[0026] In a third aspect, the present application provides a drug for inhibiting the expression of pro-inflammatory factors, wherein the drug comprises siRNA, and the nucleic acid sequence of the siRNA comprises the sequence shown in SEQ ID NO. 3 and / or SEQ ID NO. 4.

[0027] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0028] Preferably, the excipient comprises any one or a combination of at least two of carriers, wetting agents, disintegrating agents, emulsifying agents, co-solvents, solubilizing agents, osmotic pressure adjusting agents, surfactants, coating materials, coloring agents, pH adjusting agents, antioxidants, bacteriostatic agents or buffers.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The siRNA interference target sequence and siRNA provided by the present application can effectively reduce the expression level of TILRR mRNA in different types of cells and organoids, thereby partially reducing the expression of pro-inflammatory factors induced by cells / organoids in response to different inflammatory stimuli, and will not completely block the innate immune signaling pathway. The operation method is simple and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure for the TILRR expression amount after transfection of siRNA into HUVEC for 48h in Example 3.

[0032] Figure 2 Figure for the gene expression amount after stimulation of HUVEC with lipopolysaccharide in Example 4.

[0033] Figure 3 Figure showing gene expression levels after lipopolysaccharide stimulation of ASC in Example 4.

[0034] Figure 4 Figure showing gene expression levels after IL-1β stimulation of kidney organoids in Example 4. DETAILED DESCRIPTION

[0035] To further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with the examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0036] Unless otherwise specified in the examples, the techniques or conditions were carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used were conventional products that can be commercially available through regular channels.

[0037] Example 1

[0038] This example provides an siRNA for preparing a TILRR expression inhibitor. According to the siRNA design principle, an siRNA sequence with a GC content of 40-50% is selected, and the blast function of the GenBank database is used to align with the human genome sequence to ensure no homology, and two siRNAs, siTILRR-1 and siTILRR-2, are determined. The Guangzhou Aik Biotechnology Company is entrusted to synthesize them by chemical method. The nucleic acid sequence of siTILRR-1 is shown in SEQ ID NO. 3, the nucleic acid sequence of the target sequence interfered by it is shown in SEQ ID NO. 1, corresponding to the 1809-1829 nucleotide site in TILRR isoform 2 RNA. The nucleic acid sequence of siTILRR-2 is shown in SEQ ID NO. 4, and the nucleic acid sequence of the target sequence interfered by it is shown in SEQ ID NO. 2, corresponding to the 1999-2020 nucleotide site in TILRR isoform 2 RNA.

[0039] SEQ ID NO. 1: GAAGUGGUCUCAUAUUGAAUG.

[0040] SEQ ID NO. 2: CCAGGAAUGUCAACUAAGAUG.

[0041] SEQ ID NO. 3: CAUUCAAUAUGAGACCACUUCTT.

[0042] SEQ ID NO. 4: CAUCUUAGUUGACAUUCCUGGTT.

[0043] Example 2

[0044] In this embodiment, the siRNA provided in Example 1 was transfected into primary umbilical vein endothelial cells (HUVECs), human primary astrocytes (ASCs), and human iPSC-induced kidney organoids, respectively. Cells were seeded at 130,000 cells / well in 12-well plates and cultured at 37°C, 5% CO2, and 90% relative humidity for 24 hours. Cell transfection was performed using a universal DNA / siRNA transfection reagent, following the product instructions. Kidney organoids were cultured at 200-300 cells / well in 6-well plates, and transfection was performed using lipid nanoparticles (LNPs). SiRNA-LNPs were prepared and encapsulated using microfluidic technology, and then added to the organoid culture medium at a concentration of 2000 ng / mL. After 48 hours of culture, RNA was extracted for subsequent experiments.

[0045] Example 3

[0046] In this embodiment, total RNA was extracted from cells or organoids 48 h after transfection, and the content of TILRR cDNA was detected by qPCR after reverse transcription. The specific steps are as follows: Total RNA extraction: Total RNA was extracted from cells or organoids transfected with siRNA using the Trizol method; Reverse transcription: Reverse transcription was performed using HiScript III Reverse Transcriptase (Novizan); qPCR detection: qPCR was performed using an SsoAdvanced Universal SYBR Green Supermix (2X) (BioRad). The reaction volume was 10 μL, with each reaction volume containing 2 μL of cDNA template. The reaction program was a. 98℃ for 3 min, b. 98℃ for 10 s, c. 60℃ for 60 s, with 40 cycles of b and c. GAPDH was used as an internal control (NC). The nucleic acid sequences of the primers for TILRR cDNA amplification are shown in SEQ ID NO. 5–SEQ ID NO. 6 and SEQ ID NO. 7–SEQ ID NO. 8, respectively.

[0047] SEQ ID NO. 5: GGACGAGATCCCTCCAAAAT.

[0048] SEQ ID NO. 6: GGCTGTTGTCATACTTCTCATGG.

[0049] SEQ ID NO. 7: AGAGCCCTGCCTGTGGTAAC.

[0050] SEQ ID NO. 8: GAAGGGGAATGCAAGAGTGTGATA.

[0051] qPCR results as shown in Figure 1 Figure 6. siRNA of TILRR significantly reduced the expression of TILRR in HUVEC, and the expression of TILRR in ASC and human iPSCs induced kidney organoids were also significantly inhibited.

[0052] Example 4

[0053] In this example, the cells or organoids transfected for 48h were stimulated with 100ng / mL IL-1β or 1 μg / mL lipopolysaccharide (LPS) for different time, then total RNA was extracted, and the expression levels of different inflammatory factor mRNA in cells or organoids were detected by qPCR after reverse transcription. The qPCR detection method and system refer to Example 3.

[0054] Figure 2 For HUVEC stimulated with 1 μg / mL LPS for 1h or 24h, the expression of TILRR, pro-inflammatory factor CXCL8, pro-inflammatory factor TNF and EndoMT related marker gene CDH2 were all significantly reduced, indicating that the reduction of TILRR expression inhibited the expression of pro-inflammatory factors in HUVEC cells. Previous experiments found that the mRNA expression level of TNF in endothelial cells increased rapidly after 1h of LPS stimulation, and the mRNA expression level of TNF decreased several times after 3h of stimulation, and the mRNA expression level of TNF after 24h of stimulation was comparable to that before stimulation, so this experiment mainly detected the mRNA expression level of TNF after short-term inflammatory stimulation. The purpose of this experiment is to verify the effect of reducing TILRR on the expression of induced genes after cells respond to inflammatory stimulation. Previous experiments found that the expression of EndoMT marker gene CDH2 was not affected after 6h of LPS stimulation in endothelial cells, but the expression of CDH2 in endothelial cells was increased after 24h of LPS stimulation, so the results only showed the expression of CDH2 after 24h of LPS treatment.

[0055] Figure 3 For ASC transfected with siTILRR-1, the expression of pro-inflammatory factors CXCL8 and CCL2 was significantly reduced after 1h or 3h of LPS stimulation, indicating that the reduction of TILRR expression inhibited the expression of pro-inflammatory factors in ASC cells.

[0056] Figure 4 For kidney organoids stimulated with 100ng / mL IL-1β for 48h, the expression of TILRR and pro-inflammatory factor CXCL8 was significantly reduced, indicating that the reduction of TILRR expression inhibited the expression of pro-inflammatory factors in human iPSCs induced kidney organoids.

[0057] In summary, the siRNA interference target sequence and siRNA provided by the application can effectively reduce the expression level of TILRR mRNA in different types of cells and organoids, thereby reducing the expression of pro-inflammatory factors induced by different inflammatory stimuli in cells / organoids, and does not completely block the innate immune signaling pathway, the operation method is simple, and has a wide application prospect.

[0058] The applicant states that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the application, which can be easily thought of by any person skilled in the art, falls within the protection scope and disclosure scope of the application.

Claims

1. Use of siRNA for the preparation of a medicament for inhibiting inflammatory responses induced by lipopolysaccharide or IL-1 β, characterized in that, The nucleic acid sequence of the siRNA is shown in SEQ ID NO. 3 and / or SEQ ID NO.

4.

2. A medicament for inhibiting inflammatory responses induced by lipopolysaccharide or IL-1 β, characterized by, The drug comprises siRNA, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO. 3 and / or SEQ ID NO.

4.

3. The medicament according to claim 2, characterized in that, The drug further comprises a pharmaceutically acceptable excipient.

4. The medicament according to claim 3, characterized in that, The excipient comprises any one or a combination of at least two of a carrier, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

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